Role of BIN1 in epithelial cell nuclear envelope folding

Abstract

The cell nucleus is a dynamic organelle that houses and protects the genome. How the morphology of the cell nucleus is determined and regulated is incompletely understood but altered nuclear shape and mechanics are present in numerous diseases. Wrinkle-like folded phenotype of the nuclear envelope (NE) is gaining recognition as a physiological feature. The folds have been shown to traverse the nucleus and be comprised of inner and outer nuclear membranes and nuclear lamina composed of A- and B-type lamins. Nuclear lamins play a crucial role as a building block of the nuclear morphology. A-type lamins scale with tissue stiffness, mechanically tuning the cell according to environmental requirements. BIN1, a membrane remodelling protein, has been shown to play a role in nuclear positioning and morphology. Mutations in BIN1 lead to centronuclear myopathy (CNM), a disorder leading to muscle weakness caused by disordered T-tubules and abnormally located, centralized nuclei in muscle cells. In this master’s thesis, the role of BIN1 as a facilitator of NE folding was investigated. The aims of the thesis were to investigate the nuclear localization of BIN1, to analyse the effects of BIN1 function on nucleus shape, to study the influence of nuclear lamin-mediated nuclear stiffness on BIN1 interactions at the NE, and finally, to enlighten the role of BIN1 in NE folding.

To study the effects of BIN1 on epithelial nucleus shape and morphology, wild type (wt) MDCKII cells were transfected to express green fluorescent protein (GFP) -conjugated point mutated CNM-linked and tubulation-deficient BIN1 mutants K35N, D151N and R154Q. The stably expressing cells were enriched by FACS-sorting. Cell samples of the wt and the established mutant cell lines were immunostained, mounted and imaged with laser scanning confocal microscope (LSCM). The resulting images were analysed using ImageJ Fiji distribution software. The nuclei were quantified for the folded NE phenotype, area, circularity, aspect ratio, solidity, intensities of lamin A/C and BIN1, fold depth and the location of BIN1 at the NE.

The analysis showed that A-type lamins form folds on the NE that traverse the nucleus. BIN1 localized to the NE and followed the contours of lamin A/C. Intracellular localization of mutated BIN1 was more cytoplasmic (D151N), intranuclear (K35N) or both (R154Q) in comparison to that in the wt, which points to changes in the BIN1 membrane interactions. In addition, these tubulation-defected BIN1 mutations caused significant changes in the shape of the nucleus, suggesting BIN1 to have a role in the maintenance of the nuclear morphology. Detailed analysis indicated that in wt cells BIN1 was located in the immediate vicinity of the NE fold at the basal side as well as on the apical end of the fold. While the mutations did not erase the cells’ ability to fold its NE, the quantification of the folds showed decreased depth in the MDCKII-BIN1-mutant-GFP cells showing proper binding but defected membrane tubulation i.e., inward protrusion of the NE.

To conclude, the results support the hypothesis that BIN1 is involved in the NE folding and the maintenance of nuclear morphology. As BIN1 is a dimer and WT-BIN1 can dimerize with mutated BIN1, a knock-out cell line should be established and super resolution microscopy imaging should be used to further investigate the mechanistic role and of BIN1 in the NE folding dynamics.